Mechanistically diverse superfamilies provide the opportunity to understand the structural bases for divergent evolution of enzyme function. In the enolase superfamily, the reactions are initiated by abstraction of the a-proton of a carboxylate anion substrate to yield a Mg^^-stabilized enolate intermediate;the intermediate is directed to product by an appropriately located active site acid. In the RuBisCO superfamily, the reactions are initiated by abstraction of the a-proton of a ketose 1-phosphate substrate to yield a Mg^'^-stabilized enolate intermediate;the potential fate(s) of the intermediate are pooriy understood but may involve tautomerization, dehydration, oxidation, and/or carboxylation. This project describes structure/function aspects of our integrated sequence-structure-computation strategy for predicting the substrates specificities and, therefore, assigning functions of uncharacterized proteins in both superfamilies. The focus is on proteins that are encoded by operons: the enzymes that catalyze successive steps in a metabolic pathway should share conserved elements of substrate specificity, thereby facilitating identification of the functions of all of the enzymes in the pathway and, therefore, new metabolism. The project is organized in three Specific Aims:
Specific Aim 1 focuses on divergent members of the muconate lactoniziing enzyme subgroup of the enolase superfamily (Lys acid/base catalysts at the ends of the second and sixth (J-strands of the barrel domain), including 1) dipeptide epimerases that are encoded by operons that also encode homologues of dipeptidases, and 2) two novel subgroups whose members are expected to catalyze "new" reactions.
Specific Aim 2 focuses on divergent members of the mandelate racemase subgroup of the enolase superfamily (an acid/base His-Asp dyad at the ends of the seventh and sixth p-strands of the TIM-barrel domain) that are encoded by operons, with these also encoding aldolases, dehydrogenases, mutarotases, and/or kinases.
Specific Aim 3 focuses on RuBisCO-like proteins (RLPs) that are encoded by operons that also encode homologues of isomerases, aldolases, transketolases, and other aldose/ketose 5-phosphate utilizing enzymes.

Public Health Relevance

; The assignment of functions to the complete set of proteins encoded by genomes is a major problem. However, when this problem is solved, their roles in molecular, cellular, and organismal functions will be known and novel targets for specific small molecule intervention can be identified, thereby providing new approaches for therapeutic design. This Program Project is focused on developing and implementing an integrated sequence-structure-computation strategy for predicting the substrate specificities of uncharacterized proteins discovered in genome projects, thereby facilitating their functional assignment.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM071790-10
Application #
8500347
Study Section
Special Emphasis Panel (ZRG1-BCMB-D)
Project Start
Project End
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
10
Fiscal Year
2013
Total Cost
$880,083
Indirect Cost
$32,186
Name
University of Illinois Urbana-Champaign
Department
Type
DUNS #
041544081
City
Champaign
State
IL
Country
United States
Zip Code
61820
Hobbs, Merlin Eric; Williams, Howard J; Hillerich, Brandan et al. (2014) l-Galactose metabolism in Bacteroides vulgatus from the human gut microbiota. Biochemistry 53:4661-70
Akiva, Eyal; Brown, Shoshana; Almonacid, Daniel E et al. (2014) The Structure-Function Linkage Database. Nucleic Acids Res 42:D521-30
Wichelecki, Daniel J; Graff, Dylan C; Al-Obaidi, Nawar et al. (2014) Identification of the in vivo function of the high-efficiency D-mannonate dehydratase in Caulobacter crescentus NA1000 from the enolase superfamily. Biochemistry 53:4087-9
Xiang, Dao Feng; Kumaran, Desigan; Swaminathan, Subramanyam et al. (2014) Structural characterization and function determination of a nonspecific carboxylate esterase from the amidohydrolase superfamily with a promiscuous ability to hydrolyze methylphosphonate esters. Biochemistry 53:3476-85
Wichelecki, Daniel J; Vendiola, Jean Alyxa Ferolin; Jones, Amy M et al. (2014) Investigating the physiological roles of low-efficiency D-mannonate and D-gluconate dehydratases in the enolase superfamily: pathways for the catabolism of L-gulonate and L-idonate. Biochemistry 53:5692-9
Bouvier, Jason T; Groninger-Poe, Fiona P; Vetting, Matthew et al. (2014) Galactaro ?-lactone isomerase: lactone isomerization by a member of the amidohydrolase superfamily. Biochemistry 53:614-6
Ghasempur, Salehe; Eswaramoorthy, Subramaniam; Hillerich, Brandan S et al. (2014) Discovery of a novel L-lyxonate degradation pathway in Pseudomonas aeruginosa PAO1. Biochemistry 53:3357-66
Groninger-Poe, Fiona P; Bouvier, Jason T; Vetting, Matthew W et al. (2014) Evolution of enzymatic activities in the enolase superfamily: galactarate dehydratase III from Agrobacterium tumefaciens C58. Biochemistry 53:4192-203
Cummings, Jennifer A; Vetting, Matthew; Ghodge, Swapnil V et al. (2014) Prospecting for unannotated enzymes: discovery of a 3',5'-nucleotide bisphosphate phosphatase within the amidohydrolase superfamily. Biochemistry 53:591-600
Wichelecki, Daniel J; Balthazor, Bryan M; Chau, Anthony C et al. (2014) Discovery of function in the enolase superfamily: D-mannonate and d-gluconate dehydratases in the D-mannonate dehydratase subgroup. Biochemistry 53:2722-31

Showing the most recent 10 out of 105 publications